The present invention relates generally to methods and systems for examining the composition of a fluid or solid sample and more particularly to methods and systems for examining the composition of a fluid or solid sample using optical spectroscopy.
With the advent and ever-increasing use of High-Performance Liquid Chromatography (HPLC) in the chemical and biotechnology arts, the need for instrumentation capable of quickly and accurately analyzing the composition of HPLC effluent samples has heightened.
In Rev. Sci. Instrum., Vol. 50, No. 1, pp. 118-126 (Jan. 1979), Johnson et al. disclose a video fluorometer - a rapid scanning fluorescence system which uses a particular method of sample illumination and a two dimensional multichannel detector (SIT vidicon detector) to acquire fluorescence excitation and emission spectra simultaneously. More specifically, the Johnson video fluorometer comprises a xenon arc lamp, the output of which is focused onto the entrance slit of a quarter meter Ebert type monochromator equipped with interchangeable 295 1/mm gratings blazed at 250 and 400 nm. The monochromator is turned on its side so that the long axis of the entrance slit is perpendicular to the long axis of a cuvette holding a fluid sample. The exit slit and the slit holder have been removed; therefore, emerging at the exit slit plane is a 2-cm vertically dispersed polychromatic beam of radiation spanning 260 nm which is focused at 1:1 magnification onto the center of the sample cuvette. At 90 degrees to the excitation beam, the fluorescent image in the cuvette is focused at 1:1 magnification onto the entrance slit of an analyzing monochromator, which is oriented in the usual fashion with the long axis of the slit parallel to the long axis of the cuvette. Again, the exit slit and the slit holder have been removed; this results in a two dimensional image at the exit plane. The image may be represented as an Emission Excitation Matrix (EEM) whose matrix elements M.sub.ij represent the fluorescence intensity excited by wavelength i and observed at wavelength j. The EEM image is subsequently focused with 2:1 demagnification onto the target of a multichannel imaging detector.
In Analytical Chemistry, Vol. 51, No. 9, pp. 1444-1446 (Aug. 1979), Hershberger et al. disclose a sub-microliter flow-through cuvette suitable for monitoring liquid chromatographic effluents. The cuvette is based on the sheath flow principle, in which the chromatographic effluent is injected into the center of an ensheathing solvent stream but does not mix with it because laminar flow conditions are maintained. The optical volume of the cuvette is varied by adjusting the relative flow rates of sheath and sample. The response of the flow cell to a chromatographic effluent was obtained using a system comprising a laser for providing excitation radiation of one wavelength, a focusing lens for focusing the laser radiation onto the flow cell, a photomultiplier tube for measuring fluorescence intensity and a microscope for collecting the fluorescent light from the cuvette and for focusing it on the photomultiplier tube.
In Anal. Chem., Vol. 53, pp. 971-975 (1981), Hershberger et al. disclose using the above-described Johnson video fluorometer to examine effluents obtained by High-Performance Liquid Chromatography through a modified Hershberger laminar flow cell.
In Anal. Chem., Vol. 58, pp. 2831-2839 (1986), Skoropinski et al. disclose a laser videofluorometer system for real-time characterization of High-Performance Liquid Chromatographic eluate. The instrument comprises a nitrogen-laser-pumped dye laser as the excitation source and a quarter meter polychromator/microchannel plate-intensified diode array as the fluorescence detector. The dye laser cavity is tuned with a moving-iron galvanometer scanner grating drive, permitting the laser output to be changed to any wavelength in its range in less than 40 ms.
The present inventors have identified the following shortcomings in the above-described prior art systems which use a white light lamp as the excitation source: (1) The lamps typically do not excite strongly in the far UV region of the spectrum (i.e. approximately 220-300 nm) where most fluorescent compounds absorb light strongly; (2) Dispersion of lamp light is typically effected using multiple grating orders, which often result in interfering light; (3) Bandwidth inaccuracies sometimes occur due to large excitation spectral bandwidths; (4) The associated detector frequently has high interchannel crosstalk; (5) In many instances, an intensifier is located between the spectrograph and the CCD camera of the detection system, which degrades spectral resolution and/or increases interchannel crosstalk; (6) The emission spectral resolution is not always constant for all spectra of the EEM across the complete wavelength range of each spectrum; (7) Both absorption spectra and fluorescence EEM's cannot be obtained for the same sample; and (8) The spectra for the EEM's are obtained along the axis of the liquid flow; therefore, when the system is used in series with HPLC, each spectrum of a given EEM arises from a solution having a different chemical composition from its neighboring spectra.
In addition, the present inventors have identified the following shortcomings in the above-described prior art systems which use laser light as an excitation source: (1) The laser source can only generate laser beams over short wavelength ranges (30-70 nm) of a region of the spectrum at one time; consequently, the operator must intervene to change from one wavelength range to another; (2) The excitation spectral resolution is only about 2 nm; (3) Only one wavelength of laser light is generated at a time, and only one fluorescence spectrum is detected at one time; consequently, when used in series with HPLC, sequentially obtained spectra may correspond to samples of different chemical compositions; and (4) Moving parts are required to change the excitation wavelength.
Additional publications of interest include Nir et al., Laser Focus World, pp. 111-120 (Aug. 1991).